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Abstract:

The present invention is directed to topical enzymatic wound debriding
compositions with enhanced enzymatic activity. These compositions
comprise a dispersed phase comprising at least one proteolytic enzyme and
at least one hydrophilic polyol; and a continuous phase comprising a
hydrophobic base.

Claims:

1. A wound debriding composition comprising: (a) a dispersed phase
comprising a liquid hydrophilic polyol and at least one proteolytic
enzyme; and (b) a continuous phase comprising a hydrophobic base; wherein
the amount of the liquid hydrophilic polyol is within .+-.10% w/w of the
optimum amount of the liquid hydrophilic polyol.

2. The composition of claim 1, wherein the amount of liquid hydrophilic
polyol is within .+-.7% w/w of the optimum amount of the liquid
hydrophilic polyol.

3. The composition of claim 1, wherein the amount of liquid hydrophilic
polyol is within .+-.5% w/w of the optimum amount of the liquid
hydrophilic polyol.

4. The composition of claim 1, wherein the proteolytic enzyme is a
metalloprotease.

5. The composition of claim 4, wherein the metalloprotease is
collagenase.

6. The composition of claim 4, wherein the metalloprotease is
thermolysin.

7. The composition of claim 1, wherein the proteolytic enzyme is a
cysteine protease.

8. The composition of claim 7, wherein the cysteine protease is papain.

9. The composition of claim 1, wherein the proteolytic enzyme is a serine
protease.

10. The composition of claim 9, wherein the serine protease is trypsin.

11. The composition of claim 1, wherein the proteolytic enzyme is an
aspartic peptidase.

12. The composition of claim 11, wherein the aspartic peptidase is
pepsin.

13. The composition of claim 1, wherein the liquid hydrophilic polyol is
a liquid polyethylene glycol, or a liquid poloxamer, or mixtures thereof.

14. The composition of claim 1, wherein the proteolytic enzyme is
suspended in the dispersed phase.

15. The composition of claim 1, wherein the proteolytic enzyme is
dissolved in the dispersed phase.

17. The composition of claim 1, wherein the dispersed phase further
comprises a solid hydrophilic polyol.

18. The composition of claim 17, wherein the solid hydrophilic polyol is
a solid polyethylene glycol, or a solid poloxamer, or mixtures thereof.

19. The composition of claim 1, wherein the composition is a semisolid.

20. The composition of claim 1, wherein the composition is anhydrous.

21. The composition of claim 1, wherein the composition is sterile.

22. A method of treating a wound in need of debridement comprising:
applying to the wound a composition comprising a dispersed phase
comprising a liquid hydrophilic polyol, and an effective debriding
concentration of at least one proteolytic enzyme; and a continuous phase
comprising a hydrophobic base; wherein the amount of liquid hydrophilic
polyol is within .+-.10% w/w of the optimum amount.

23. (canceled)

24. A method of determining an optimum amount of liquid hydrophilic
polyol to add to a target composition comprising a dispersed phase
including a proteolytic enzyme and a continuous phase including a
hydrophobic base, the method comprising: (1) obtaining a series of
compositions comprising the dispersed phase and the continuous phase,
wherein the dispersed phase further includes a liquid hydrophilic polyol,
and wherein each composition in the series of compositions include an
identical amount of proteolytic enzyme and a different amount of the
liquid hydrophilic polyol; (2) determining the enzymatic activity of each
composition in the series of compositions; (3) determining the highest
point on a graph that plots the enzymatic activity versus the amount of
liquid hydrophilic polyol(s) included in each composition of the series
of compositions, wherein the highest point on the graph correlates to the
optimum amount of liquid hydrophilic polyol to add to the target
composition.

25.-30. (canceled)

31. A method of increasing enzymatic activity in a target composition
comprising a dispersed phase including a proteolytic enzyme and a
continuous phase including a hydrophobic base, the method comprising: (1)
obtaining a series of compositions comprising the dispersed phase and the
continuous phase, wherein the dispersed phase further includes a liquid
hydrophilic polyol, and wherein each composition in the series of
compositions includes an identical amount of proteolytic enzyme and a
different amount of the liquid hydrophilic polyol; (2) determining the
enzymatic activity of each composition in the series of compositions; (3)
determining the highest point on a graph that plots the enzymatic
activity versus the amount of liquid hydrophilic polyol(s) included in
each composition of the series of compositions, wherein the highest point
on the graph correlates to an optimum amount of liquid hydrophilic polyol
to add to the target composition, and (4) adding .+-.10% w/w of the
optimum amount of liquid hydrophilic polyol to the target composition,
thereby increasing the enzymatic activity in the target composition.

32.-36. (canceled)

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application
No. 61/267,730, filed 8 Dec. 2009. The contents of the referenced
application are incorporated by reference.

[0005] The healing of wounds is a complex process which is often further
complicated by the presence of non-viable, necrotic tissue in the wound
area. Debridement is the process of removing the non-viable tissue from a
wound to prevent or diminish infection and facilitate healing. Topical
compositions containing proteolytic enzymes such as trypsin, papain,
bromelain, subtilisin, sutilains, and collagenase have been used for
enzymatic wound debridement. Generally, the standard of care is to apply
the composition to the wound in need of debridement once daily (once
every 24 hours) or more often if the composition becomes soiled. Because
many proteolytic enzymes are susceptible to degradation in water-based
compositions, many wound debriding compositions are made with anhydrous,
hydrophobic bases such as petrolatum, mineral oil and/or vegetable oil as
disclosed in U.S. Pat. No. 3,821,364 and U.S. Pat. No. 6,479,060, both of
which are herein incorporated by reference. However, enzymatic wound
debriding compositions based on hydrophobic bases are generally not
miscible in the aqueous environment of a wound bed, and thus contact of
the proteolytic enzyme with the wound bed is generally hindered. Some
other compositions are made with anhydrous, hydrophilic bases such as
propylene glycol or poloxamers as disclosed in U.S. Pat. No. 6,548,556,
US 2003/0198631 and US 2003/0198632, all of which are herein incorporated
by reference.

SUMMARY OF THE INVENTION

[0006] The present invention is directed to topical enzymatic wound
debriding compositions with enhanced enzymatic activity. These
compositions comprise a dispersed phase comprising at least one
proteolytic enzyme and at least one hydrophilic polyol; and a continuous
phase comprising a hydrophobic base. The wound debriding compositions of
the present invention possess enhanced enzymatic activity over wound
debriding compositions of the prior art.

[0007] In one aspect of the present invention, there is disclosed a wound
debriding composition comprising a dispersed phase comprising a liquid
hydrophilic polyol and at least one proteolytic enzyme; and a continuous
phase comprising a hydrophobic base; wherein the amount of liquid
hydrophilic polyol is within ±10% w/w of the optimum amount of the
liquid hydrophilic polyol. For example, if the optimum amount was about
30% w/w, the amount of liquid hydrophilic polyol that could be used would
be between about 20% w/w and about 40% w/w of the total formulation to
achieve enhanced enzymatic activity of the formulation. In another
aspect, the amount of liquid hydrophilic polyol is within ±9%, 8%, 7%,
or 6% w/w of the optimum amount of the liquid hydrophilic polyol. In
still another aspect, the amount of liquid hydrophilic polyol is within
±5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% w/w of the optimum amount of the
liquid hydrophilic polyol.

[0008] The "optimum amount of liquid hydrophilic polyol" in a composition
comprising (a) a dispersed phase including a liquid hydrophilic polyol
and at least one proteolytic enzyme; and (b) a continuous phase
comprising a hydrophobic base can be determined by the method described
in Section A of the Detailed Description section of this specification,
which is incorporated into this section by reference.

[0009] A method for determining whether a composition is within +10% w/w
of the optimum amount of a liquid hydrophilic polyol is described in
Section B of the Detailed Description section of this specification,
which is incorporated by reference.

[0010] The optimum amount of liquid hydrophilic polyol for compositions
with different proteolytic enzymes can differ. Additionally, the optimum
amount of liquid hydrophilic polyol for compositions with a specific
proteolytic enzyme can differ depending on the ingredients of the
composition. For example, the optimum amount of liquid hydrophilic polyol
in a collagenase composition containing PEG-400 and petrolatum can be
different from the optimum amount of liquid hydrophilic polyol in a
collagenase composition containing PEG-600 and petrolatum, or different
from a collagenase composition containing poloxamer-124 and petrolatum.

[0011] The term "hydrophilic polyol" means water-soluble, polar aliphatic
alcohols with at least two hydroxyl groups and includes, but is not
limited to, polymeric polyols (e.g., polyethylene glycols and
poloxamers).

[0012] The term "liquid" in the context of describing "hydrophilic
polyol", "polyethylene glycol", or "poloxamer" means that the material is
in the liquid state at 25° C.

[0013] The term "solid" in the context of describing "hydrophilic polyol",
"polyethylene glycol", or "poloxamer" means that the material is in the
solid state at 25° C.

[0014] In another aspect of the present invention, there is disclosed a
method of treating a wound in need of debridement comprising: applying to
the wound a composition comprising a dispersed phase comprising a liquid
hydrophilic polyol, and an effective debriding concentration of at least
one proteolytic enzyme; and a continuous phase comprising a hydrophobic
base; wherein the amount of liquid hydrophilic polyol is within ±10%
w/w of the optimum amount. In another aspect, the amount of liquid
hydrophilic polyol is within ±9%, 8%, 7%, or 6% w/w of the optimum
amount. In still another aspect, the amount of liquid hydrophilic polyol
is within ±5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% w/w of the optimum
amount.

[0015] In some embodiments, the proteolytic enzyme is a metalloprotease, a
cysteine protease, a serine protease, or an aspartic peptidase.
Generally, the optimum amount of hydrophilic polyol for compositions
comprising a metalloprotease, a cysteine protease or a serine protease is
from about 10%, 11%, 12%, 13%, 14%, 15%, 16% 17%, 18%, 19%, 20%, 21%,
22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,
36%, 37%, 38%, 39% w/w to about 40% w/w, or any range or numerical amount
derivable therein. The optimum amount of hydrophilic polyol for
compositions comprising an aspartic peptidase is from about 48%, 49%,
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
64%, 65%, 66%, 67% w/w to about 68% w/w or any range or numerical amount
derivable therein. In one embodiment the metalloprotease is collagenase.
In another embodiment the metalloprotease is collagenase and the optimum
amount of the hydrophilic polyol is from about 10%, 11%, 12%, 13%, 14%,
15%, 16% 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% w/w to about 40% w/w or
any range or numerical amount derivable therein. In one embodiment, the
metalloprotease is thermolysin. In another embodiment, the
metalloprotease is thermolysin and the optimum amount hydrophilic polyol
is from about 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,
31%, 32%, 33%, 34%, 35%, 36%, 37%, 38% w/w to about 39% w/w or any range
or numerical amount derivable therein. In one embodiment, the cysteine
protease is papain. In another embodiment the cysteine protease is papain
and the optimum amount of the hydrophilic polyol is from about 19%, 20%,
21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,
35%, 36%, 37%, 38% w/w to about 39% w/w or any range or numerical amount
derivable therein. In one embodiment, the serine protease is trypsin. In
another embodiment the serine protease is trypsin and the optimum amount
of hydrophilic polyol is from about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%, 14%, 15%, 16% 17%, 18%, 19%, 20%, 21%, 22%, 23% w/w to about
24% w/w or any range or numerical derivable therein. In one embodiment,
the aspartic peptidase is pepsin. In another embodiment the aspartic
peptidase is pepsin and the optimum amount of hydrophilic polyol is from
about 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67% w/w to about 68% w/w or any range or
numerical amount derivable therein. In some embodiments, the proteolytic
enzyme is suspended in the dispersed phase. In other embodiments the
proteolytic enzyme is dissolved in the dispersed phase.

[0016] In some embodiments, the liquid hydrophilic polyol is a liquid
polyethylene glycol or a liquid poloxamer, or mixtures thereof.

[0017] In some embodiments of the present invention, the dispersed phase
may further comprise a solid hydrophilic polyol in order to help
physically stabilize the composition or reduce or prevent phase
separation. In some embodiments, the solid hydrophilic polyol is a solid
poloxamer, or a solid polyethylene glycol, or mixtures thereof.

[0018] In various embodiments of the present invention, the hydrophobic
base comprises petrolatum, mineral oil, or vegetable oil, or mixtures
thereof. In one embodiment, the base comprises petrolatum. In another
embodiment, the hydrophobic base comprises a vegetable oil. In still
another embodiment, the hydrophobic base comprises mineral oil. In a
further embodiment, the hydrophobic base comprises petrolatum and mineral
oil, petrolatum and vegetable oil, mineral oil and vegetable oil, or
petrolatum, mineral oil, and vegetable oil.

[0019] In still another embodiment, the hydrophobic base comprises a
vegetable oil, wherein the vegetable oil is castor oil.

[0020] In one embodiment, the composition is a semisolid. In another
embodiment, the composition is a liquid. In other embodiments, the
composition is impregnated on a pad, gauze, or sponge. In one embodiment,
the composition is sterile or anhydrous or both.

[0022] In another embodiment of the present invention there is disclosed a
method of determining the optimum amount of liquid hydrophilic polyol to
add to a target composition comprising a dispersed phase including a
proteolytic enzyme and a continuous phase including a hydrophobic base,
the method comprising: (1) obtaining a series of compositions comprising
the dispersed phase and the continuous phase, wherein the dispersed phase
further includes a liquid hydrophilic polyol, and wherein each
composition in the series of compositions include an identical amount of
proteolytic enzyme and a different amount of the liquid hydrophilic
polyol; (2) determining the enzymatic activity of each composition in the
series of compositions; (3) determining the highest point on a graph that
plots the enzymatic activity versus the amount of liquid hydrophilic
polyol(s) included in each composition of the series of compositions,
wherein the highest point on the graph correlates to the optimum amount
of liquid hydrophilic polyol to add to the target composition. In one
aspect, the enzymatic activity of the series of compositions can be
determined by using the in-vitro artificial eschar testing model as
described in this specification.

[0023] In a further aspect of the present invention there is disclosed a
method of increasing enzymatic activity in a target composition
comprising a dispersed phase including a proteolytic enzyme and a
continuous phase including a hydrophobic base, the method comprising: (1)
obtaining a series of compositions comprising the dispersed phase and the
continuous phase, wherein the dispersed phase further includes a liquid
hydrophilic polyol, and wherein each composition in the series of
compositions includes an identical amount of proteolytic enzyme and a
different amount of the liquid hydrophilic polyol; (2) determining the
enzymatic activity of each composition in the series of compositions; (3)
determining the highest point on a graph that plots the enzymatic
activity versus the amount of liquid hydrophilic polyol(s) included in
each composition of the series of compositions, wherein the highest point
on the graph correlates to an optimum amount of liquid hydrophilic polyol
to add to the target composition, and (4) adding ±10% w/w of the
optimum amount of liquid hydrophilic polyol to the target composition,
thereby increasing the enzymatic activity in the target composition. In
one aspect, the enzymatic activity of the series of compositions can be
determined by using the in-vitro artificial eschar testing model as
described in this specification.

[0025] The term "anhydrous" means that the compositions contain less than
about 5% w/w, or less than about 3% w/w, or less than about 1% w/w, or
less than about 0.5% w/w, or less than about 0.1% w/w in relation to the
total composition, or 0%, of free or added water, not counting the water
of hydration, bound water, or typical moisture levels present in any of
the raw ingredients of the compositions.

[0026] Unless otherwise specified, the percent values expressed herein are
weight by weight and are in relation to the total composition.

[0027] The use of the word "a" or "an" when used in conjunction with the
term "comprising" or "containing" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one or
more," "at least one," and "one or more than one."

[0028] Throughout this application, the term "about" is used to indicate
that a value includes the inherent variation of error for the device
obtaining the value, the method being employed to determine the value, or
the variation that exists among the objects being evaluated.

[0029] As used in this specification and claim(s), the words "comprising"
(and any form of comprising, such as "comprise" and "comprises"),
"having" (and any form of having, such as "have" and "has"), "including"
(and any form of including, such as "includes" and "include") or
"containing" (and any form of containing, such as "contains" and
"contain") are inclusive or open-ended and do not exclude additional,
unrecited elements or method steps.

[0030] The terms "treating," "inhibiting," "preventing, or "reducing" or
any variation of these terms, when used in the claims and/or the
specification includes any measurable decrease or complete inhibition to
achieve a desired result.

[0031] The term "effective," as that term is used in the specification
and/or claims, means adequate to accomplish a desired, expected, or
intended result.

[0032] The compositions and methods for their use can "comprise," "consist
essentially of," or "consist of" any of the ingredients or steps
disclosed throughout the specification. With respect to the transitional
phase "consisting essentially of," and in one non-limiting aspect, a
basic and novel characteristic of the compositions and methods disclosed
in this specification includes the composition's enhanced enzymatic
activity.

[0033] Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It should
be understood, however, that the detailed description and the specific
examples, while indicating specific embodiments of the invention, are
given by way of illustration only, since various changes and
modifications within the spirit and scope of the invention will become
apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1. Plot of the in-vitro collagenolysis activity (mg/ml) of a
series of compositions comprising a dispersed phase comprising
collagenase and PEG-400, dispersed in a hydrophobic phase comprising
white petrolatum (y-axis) versus the percentage of the PEG-400 comprised
in the series of compositions x-axis).

[0035] FIG. 2. Plot of the in-vitro collagenolysis activity (mg/ml) of a
series of compositions comprising a dispersed phase comprising
collagenase and PEG-600, dispersed in a hydrophobic phase comprising
white petrolatum (y-axis) versus the percentage of the PEG-600 comprised
in the series of compositions x-axis).

[0036] FIG. 3. Plot of the in-vitro collagenolysis activity (mg/ml) of a
series of compositions comprising a dispersed phase comprising
collagenase and poloxamer-124, dispersed in a hydrophobic phase
comprising white petrolatum (y-axis) versus the percentage of the
poloxamer-124 comprised in the series of compositions x-axis).

[0037] FIG. 4. Plot of the in-vitro collagenolysis activity (mg/ml) of a
series of compositions comprising a dispersed phase comprising trypsin
and PEG-400, dispersed in a hydrophobic phase comprising white petrolatum
(y-axis) versus the percentage of the PEG-400 comprised in the series of
compositions x-axis).

[0038] FIG. 5. Plot of the in-vitro collagenolysis activity (mg/ml) of a
series of compositions comprising a dispersed phase comprising papain and
PEG-400, dispersed in a hydrophobic phase comprising white petrolatum
(y-axis) versus the percentage of the PEG-400 comprised in the series of
compositions x-axis).

[0039]FIG. 6. Plot of the in-vitro collagenolysis activity (mg/ml) of a
series of compositions comprising a dispersed phase comprising
thermolysin and PEG-400, dispersed in a hydrophobic phase comprising
white petrolatum (y-axis) versus the percentage of the PEG-400 comprised
in the series of compositions x-axis).

[0040] FIG. 7. Plot of the in-vitro collagenolysis activity (mg/ml) of a
series of compositions comprising a dispersed phase comprising pepsin and
PEG-400, dispersed in a hydrophobic phase comprising white petrolatum
(y-axis) versus the percentage of the PEG-400 comprised in the series of
compositions x-axis).

[0041] FIG. 8. Plot of the physical release of collagenase (mg) from of a
series of compositions comprising a dispersed phase comprising
collagenase and PEG-400, dispersed in a hydrophobic phase comprising
white petrolatum (y-axis) versus the percentage of the PEG-400 comprised
in the series of compositions x-axis).

[0044] One aspect of the present invention provides for topical enzymatic
wound debriding compositions with enhanced enzymatic activity. These
compositions comprise a dispersed phase comprising at least one
proteolytic enzyme and a hydrophilic polyol; and a continuous phase
comprising a hydrophobic base. In one aspect of the invention, the
hydrophilic polyol is a liquid hydrophilic polyol.

[0045] It was found that the enzymatic activity (e.g., in vitro
collagenolysis) of the compositions of the present invention, which are
dispersions of a hydrophilic polyol and a proteolytic enzyme in a
hydrophobic base, not only was higher than the enzymatic activity of
enzyme compositions based solely on a proteolytic enzyme and hydrophobic
base combination (i.e., no hydrophilic phase such as a hydrophilic
polyol), but also surprisingly higher than those enzyme compositions
based solely on a proteolytic enzyme and hydrophilic base combination
(i.e., no hydrophobic phase such as petrolatum). Since enzymes are
activated in the presence of moisture, it would have been expected to see
the highest enzymatic activity in compositions based solely on a
proteolytic enzyme and hydrophilic base combination, where the base would
be completely miscible in moisture and conditions would be the most
favorable for release and activation of the enzyme. However, the
dispersion composition of hydrophilic and hydrophobic phases of the
present invention had the highest enzymatic activity correlating to an
optimum amount of the hydrophilic polyol which was more than 0% and less
than 100% of the hydrophilic polyol in the composition.

[0046] It was found, expectedly, that the physical enzyme release in
compositions based solely on a hydrophilic vehicle was greater than the
release of the enzyme in compositions based solely on a hydrophobic
vehicle, and also more than compositions of the present invention. As
seen in FIG. 8, the enzyme release profile generally increased with the
increasing percentage of hydrophilic polyol (PEG-400), with the highest
release at 100% and the lowest release at 0%. However, surprisingly, the
enzymatic activity was greater with the dispersion compositions of the
present invention (see FIGS. 1-7). Thus the enzymatic activity profile of
these dispersion compositions does not correlate with the physical enzyme
release profile as would be expected.

[0047] The compositions of the present invention are suitable for
treatment of a wound in need of debridement by applying to the wound a
composition comprising a dispersed phase comprising a hydrophilic polyol,
and an effective debriding concentration of at least one proteolytic
enzyme; and a continuous phase comprising a hydrophobic base; wherein the
amount of hydrophilic polyol is within ±10% w/w of the optimum amount,
or ±9%, 8%, 7%, or 6% w/w of the optimum amount, or ±5%, 4%, 3%,
2%, 1%, 0.5%, or 0.1% w/w of the optimum amount of hydrophilic polyol.

[0048] These and other non-limiting aspects of the present invention are
discussed in further detail in the following sections.

[0049] The following protocol can be used to prepare a series of
compositions (referred to as "Series of Compositions") and to
subsequently determine the optimum amount of liquid hydrophilic polyol
that can be used in a dispersion of the present invention:

[0050] Eleven (11) compositions can be used to create the Series of
Compositions. Note that the amount (% w/w) of proteolytic enzyme in the
series of compositions is held constant. The following steps can be used
to prepare the eleven (11) compositions:

[0051] (i) Determine the ingredients (i.e., liquid hydrophilic polyol,
proteolytic enzyme, and hydrophobic base) to be used in the Series of
Compositions and select the amount of proteolytic enzyme to be used. By
way of example, liquid hydrophilic polyol (e.g., PEG 400), proteolytic
enzyme (e.g., collagenase at 1% w/w), and hydrophobic base (e.g., white
petrolatum).

[0052] (ii) For composition one in the Series of Compositions, use 0% of
the liquid hydrophilic polyol, use the selected amount of proteolytic
enzyme, and q.s the batch with the hydrophobic base to 100%. For example,
and referring to step (i) above, composition one of the Series of
Compositions would have: 0% w/w PEG 400, 99% w/w of white petrolatum, and
1% w/w of collagenase.

[0053] (iii) For composition two in the Series of Compositions, use 10%
w/w of the liquid hydrophilic polyol, the same amount of the proteolytic
enzyme, and q.s. the batch with the hydrophobic base to 100%. (Note that
it is permissible to use some solid hydrophilic polyol in the makeup of
the liquid hydrophilic polyol as necessary to produce a physically stable
dispersion for compositions in the Series of Compositions).

[0054] (iv) For composition three in the Series of Compositions, use 20%
w/w of the liquid hydrophilic polyol, the same amount of the proteolytic
enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0055] (v) For composition four in the Series of Compositions, use 30% w/w
of the liquid hydrophilic polyol, the same amount of the proteolytic
enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0056] (vi) For composition five in the Series of Compositions, use 40%
w/w of the liquid hydrophilic polyol, the same amount of the proteolytic
enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0057] (vii) For composition six in the Series of Compositions, use 50%
w/w of the liquid hydrophilic polyol, the same amount of the proteolytic
enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0058] (viii) For composition seven in the Series of Compositions, use 60%
w/w of the liquid hydrophilic polyol, the same amount of the proteolytic
enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0059] (ix) For composition eight in the Series of Compositions, use 70%
w/w of the liquid hydrophilic polyol, the same amount of the proteolytic
enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0060] (x) For composition nine in the Series of Compositions, use 80% w/w
of the liquid hydrophilic polyol, the same amount of the proteolytic
enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0061] (xi) For composition ten in the Series of Compositions, use 90% w/w
of the liquid hydrophilic polyol, the same amount of the proteolytic
enzyme, and q.s. the batch with the hydrophobic base to 100%.

[0062] (xii) For composition eleven in the Series of Compositions, use 0%
of the hydrophobic base, the same amount of the proteolytic enzyme, and
q.s. the batch with the hydrophilic polyol.

[0063] (xiii) determine the enzymatic activity of each of the eleven
compositions in the Series of Compositions by using the in vitro
artificial eschar testing model for the following sample collection
times: 6, 12, 18 and 24 hours, as described in Section H of the Detailed
Description section of this specification.

[0064] (ivx) plot a curve of the enzymatic activity of each composition
versus the correlating amount of liquid hydrophilic polyol(s) present in
each composition of the Series of Compositions cumulatively for each data
collection time. The highest point on the curve for the cumulative
24-hour data collection time correlates to the optimum amount of liquid
hydrophilic polyol that can be used in a dispersion.

[0065] Further, given that multiple ingredients can be included in the
Series of Compositions (e.g., polyol(s) proteolytic enzyme(s),
hydrophobic base, and additional ingredients within the dispersed phase,
and/or additional ingredients within the continuous hydrophobic phase),
the Series of Compositions can be created by (1) varying the amount of
hydrophilic polyol as discussed above for each composition in the series,
(2) using the determined amount of proteolytic enzyme, and (3) q.s.-ing
the batch to 100% with the amount of the additional ingredients including
the hydrophobic base; except for composition eleven, where the batch
would be q.s.-ed to 100% with the amount of the additional ingredients
including the hydrophilic polyol.

B. Method for Determining Whether a Composition has +/-10% w/w of the
Optimum Amount of Liquid Hydrophilic Polyol

[0066] It can be determined if a composition comprising (a) a dispersed
phase including a liquid hydrophilic polyol and at least one proteolytic
enzyme; and (b) a continuous phase comprising a hydrophobic base
(referred to as "Composition of Interest") is within +10% of the Optimum
Amount of liquid hydrophilic polyol by using the following protocol:

[0067] Step One: Obtain a Composition of Interest that includes: (i) a
dispersed phase including a liquid hydrophilic polyol(s) and a
proteolytic enzyme and (ii) a continuous phase including a hydrophobic
base.

[0068] Step Two: Prepare a series of compositions (referred to as "Series
of Compositions") based on the Composition of Interest. Note that the
amount (% w/w) of proteolytic enzyme in the Series of Compositions is
held constant and is the same as the amount (% w/w) present in the
Composition of Interest. The following steps can be used to prepare the
Series of Compositions:

[0069] (i) Determine the amount of all ingredients in the Composition of
Interest (% w/w).

[0070] (ii) Determine the total amount of the continuous phase in the
Composition of Interest (% w/w). By way of example, if the Composition of
Interest includes 15% w/w liquid hydrophilic polyol (e.g., PEG 400), 1%
w/w proteolytic enzyme (e.g., collagenase), and 84% w/w hydrophobic base
(e.g., white petrolatum), then the Composition of Interest would be 84%
w/w continuous phase and 16% w/w dispersed phase.

[0071] Step Three: Prepare the Series of Compositions in a manner
described above in Section A of this specification (e.g., this would
include preparing 11 compositions in a manner described in Section A of
this specification).

[0072] Step Four: Determine the enzymatic activity of each of the eleven
compositions in the Series of Compositions by using the in vitro
artificial eschar testing model for each of the following sample
collection times: 6, 12, 18 and 24 hours as described in Section H of the
Detailed Description section of this specification.

[0073] Step Five: Plot a curve of the enzymatic activity of each
composition versus the correlating amount of liquid hydrophilic polyol(s)
present in each composition of the Series of Compositions cumulatively
for each data collection time. The highest point on the curve for the
cumulative 24-hour data collection time correlates to the optimum amount
of liquid hydrophilic polyol for the Composition of Interest.

[0074] Step Six: Compare the amount of liquid hydrophilic polyol present
within the Composition of Interest to determine whether it is within
±10% w/w of the optimum amount of liquid hydrophilic polyol for the
Composition of Interest.

C. Proteolytic Enzymes

[0075] Any proteolytic enzyme useful for wound debridement is suitable for
the present invention. Proteolytic enzymes (proteases) break down protein
by hydrolysis of the peptide bonds that link amino acids together in the
polypeptide chain of a protein. They are divided into four major groups
on the basis of catalytic mechanism: serine proteases, cysteine
proteases. metalloproteases, and aspartic proteases. Some proteases have
been identified with other catalytic amino acids in the active site, such
as threonine and glutamic acid; however, they do not form major groups.

[0076] 1. Serine Proteases

[0077] Serine proteases depend upon the hydroxyl group of a serine residue
acting as the nucleophile that attacks the peptide bond. The major clans
found in humans include the chymotrypsin-like, the subtilisin-like, the
alpha/beta hydrolase, and signal peptidase clans. In evolutionary
history, serine proteases were originally digestive enzymes. In mammals,
they evolved by gene duplication to serve functions in blood clotting,
the immune system, and inflammation. These proteases have a broad
substrate specificity and work in a wide pH range. Non-limiting examples
of serine proteases include trypsin, chymotrypsin, subtilisin, sutilains,
plasmin, and elastases.

[0078] 2. Cysteine Proteases

[0079] Peptidases in which the nucleophile that attach the scissile
peptide bond in the sulfhydryl group of a cysteine residue are known as
cysteine proteases. Cysteine proteases are commonly encountered in fruits
including papaya, pineapple, and kiwifruit. Cysteine proteases have a
broad specificity and are widely used under physiological conditions. In
this family, papain has been used extensively for wound debridement for a
long time. Other cysteine proteases, such as bromelain and analain, have
also been investigated for the applications in wound debridement. Other
non-limiting examples of cysteine proteases include calpain, caspases,
chymopapain, and clostripain.

[0080] 3. Metalloproteases

[0081] Metalloproteases are among the proteases in which the nucleophilic
attach on a peptide bond is mediated by a water molecule, while a
divalent metal cation, usually zinc but sometimes cobalt, manganese,
nickel or copper, activates the water molecule. The metal ions are
extremely important for the activity. Any compounds that have potential
to interact with the metal ion, chelating or oxidation, will affect the
enzymatic activity. Non-limiting examples of metalloproteases in this
family include thermolysin, collagenases, matrix metallo proteinases
(MMPs), bacillolysin, dispase, vibriolysin, pseudolysin, stromelysin, and
various bacterial derived neutral metalloproteases.

[0082] 4. Aspartic Peptidases

[0083] Aspartic peptidases are so named because aspartic acid residues are
the ligands of the activated water molecule. In most enzymes in this
family, a pair of aspartic residues act together to bind and activate the
catalytic water molecule. All or most aspartic peptidases are
endopeptidases. Most aspartic peptidases have a broad specificity.
However, the optimum pH of most aspartic peptidases is in the acidic
range. Non-limiting examples of aspartic peptidases are pepsin, chymosin,
beta-secretase, plasmepsin, plant acid proteases and retroviral
proteases.

[0084] 5. Collagenase

[0085] A suitable proteolytic enzyme for wound debridement is the
metalloprotease collagenase. The collagenase can be substantially pure or
it may contain detectable levels of other proteases.

[0086] The potency assay of collagenase, and meaning of "collagenase
units" as used herein, is based on the digestion of undenatured collagen
from (bovine Achilles tendon) at pH 7.2 and 37° C. for 24 hours.
The number of peptide bonds cleaved is measured by reaction with
ninhydrin. Amino groups released by a trypsin digestion control are
subtracted. One net collagenase unit will solubilize ninhydrin reactive
material equivalent to 1 nanomole of leucine equivalents per minute.

[0089] In one embodiment, the collagenase is derived from Clostridium
histolyticum; however, in other embodiments the collagenase can be
derived from other sources. Methods for producing a suitable collagenase
are disclosed in U.S. Pat. Nos. 3,705,083; 3,821,364; 5,422,261;
5,332,503; 5,422,103; 5,514,370; 5,851,522; 5,718,897; and 6,146,626 all
of which are herein incorporated by reference.

[0090] 6. Trypsin

[0091] Another suitable proteolytic enzyme for wound debridement is the
serine protease trypsin. Typically, trypsin is derived from the pancreas
of healthy bovine or porcine animals, or both. Trypsin can also be
derived from recombinant sources. The pharmaceutical grade (USP/NF) of
trypsin is known as Crystallized Trypsin. It contains not less than 2500
USP Trypsin Units per mg, calculated on the dried basis, and not less
than 90.0% and not more than 110.0% of the labeled potency. The potency
assay of trypsin as well as the definition of a USP Trypsin Unit are
found in the Crystallized Trypsin monograph of the USP 31 (Official Aug.
1, 2008) herein incorporated by reference.

[0092] The amount (potency or concentration) of trypsin in the
compositions of the present invention is at an effective level to debride
the wound. Generally, the potency of trypsin in the compositions can vary
from about 90 to about 60,000 USP Trypsin Units per gram of product. In
various embodiments the potency of trypsin, expressed as USP Trypsin
Units per gram of product, is from about 90, 100, 150, 200, 250, 300,
320, 350, 375, 400, 500, 600, 675, 700, 800, 900, 1000, 2000, 3000, 4000,
5000, 10000, 20000, 30000, 40000, 50000 to about 60000, or any range or
numerical amount derivable therein.

[0094] Hydrophilic polyols of the present invention are water-soluble,
polar aliphatic alcohols with at least two hydroxyl groups, and include
polymeric polyols, e.g., polyethylene glycols and poloxamers. In one
aspect of the invention, the hydrophilic polyol in the dispersed phase is
a liquid hydrophilic polyol. In some embodiments, the liquid hydrophilic
polyol is a liquid polyethylene glycol or a liquid poloxamer, or mixtures
thereof. Solid hydrophilic polyols such as solid polyethylene glycols or
solid poloxamers can also be added to the dispersed phase of the
invention to help physically stabilize the dispersion. Other examples of
liquid hydrophilic polyols include but are not limited to propylene
glycol, butylene glycol, pentylene glycol, hexylene glycol, glycerin,
hexylene glycol, methoxy polyethylene glycol, propylene carbonate, and
ethoxydiglycol, and these may also be added to the dispersed phase.

[0095] 1. Polyethylene Glycols

[0096] Polyethylene glycols are homo-polymers of ethylene glycol and water
represented by the formula:

H(OCH2CH2)nOH

in which n represents the average number of oxyethylene groups.
Polyethylene glycols can be either liquid or solid at 25° C.
depending on their molecular weights.

[0099] The liquid and solid polyethylene glycols are available
commercially from the DOW Chemical Company under the CARBOWAX®
tradename and from the BASF Corporation under LUTROL® E and
PLURACARE® E tradenames. Both pharmaceutical grade (USP/NF) and
cosmetic grade polyethylene glycols are suitable for the present
invention.

[0100] 2. Poloxamers

[0101] Poloxamers are synthetic block copolymers of ethylene oxide and
propylene oxide represented by the formula:

HO(C2H4O).sub.a(C3H6O).sub.b(C2H4O).sub.aH

in which formula a and b represent the number of repeat units. Generally
a is from 2 to 150 and b is from 15 to 70 depending on the particular
poloxamer. Poloxamers can be either liquid or solid at 25° C.
depending on their molecular weights.

[0104] The liquid and solid poloxamers are available commercially from the
BASF Corporation under the PLURONIC® and LUTROL® tradenames and
from the UNIQEMA Corporation under the SYNPERONIC® trademark.
Pharmaceutical grade (USP/NF) poloxamers are poloxamer 124, poloxamer
188, poloxamer 237, poloxamer 338, and poloxamer 407. Both pharmaceutical
grade and cosmetic grade poloxamers are suitable for the present
invention.

E. Hydrophobic Bases

[0105] The hydrophobic bases of the present invention can comprise, but
are not limited to, plant, animal, paraffinic, and synthetic derived
fats, butters, greases, waxes, solvents, and oils; mineral oils,
vegetable oils, petrolatum, water insoluble organic esters and
triglycerides, silicones, or fluorinated compounds; or mixtures thereof.
In one embodiment of the present invention the hydrophobic phase
comprises petrolatum.

[0110] Non-limiting examples of silicones are dimethicone and
cyclomethicone. A non-limiting example of a fluorinated compound is
polytetrafluoroethylene (PTFE).

[0111] 1. Petrolatum

[0112] Petrolatum is a purified mixture of semisolid hydrocarbons obtained
from petroleum and varies from dark amber to light yellow in color. White
petrolatum is wholly or nearly decolorized petrolatum and varies from
cream to snow white in color. Petrolatum and White Petrolatum can also
vary in melting point, viscosity, and consistency.

[0114] Petrolatum and White Petrolatum are available in cosmetic grade and
pharmaceutical (USP/NF) grade and both are suitable for the present
invention.

F. Topical Compositions

[0115] The topical compositions of the present invention are dispersions
comprising a hydrophilic dispersed phase in a hydrophobic continuous
phase. The dispersed phase comprises a proteolytic enzyme and a
hydrophilic polyol. In an aspect of the invention, the hydrophilic polyol
is a liquid hydrophilic polyol. In some embodiments, the liquid
hydrophilic polyol is a liquid polyethylene glycol or a liquid poloxamer,
or mixtures thereof. The continuous phase comprises a hydrophobic base.
The hydrophobic base can be petrolatum. The compositions are useful for
treatment of wounds for wound debridement.

[0116] The compositions can be anhydrous as defined herein. The
compositions can be semisolid or liquid. The composition can be
impregnated on a pad, gauze, or sponge. The compositions can also be
sterile.

[0117] The compositions can include additional materials known in the art
that are suitable for topical compositions of this nature, e.g.,
absorbents, deodorizers, surfactants, solvents, rheology modifiers, film
formers, stabilizers, emollients, moisturizers, preservatives,
antimicrobials, antioxidants, chelating agents, fragrances, and
colorants.

[0118] The compositions can also include additional pharmaceutical active
ingredients known in the art that are suitable for topical compositions
of this nature, e.g., antimicrobial agents, wound healing agents,
anesthetic agents, vulnerary agents, and haemostatic agents. A
non-limiting example of a vulnerary agent is balsam Peru.

[0120] The compositions of the present invention can be prepared by
techniques and methods known by one of ordinary skill in the art by
dissolving or suspending the proteolytic enzyme in part or all of the
available hydrophilic polyol. The resulting solution or suspension can be
mixed with a hydrophobic base to form a dispersion, wherein the
hydrophobic base becomes the continuous phase and the hydrophilic
polyol/enzyme phase becomes the dispersed phase. These compositions can
be prepared using processing equipment known by one of ordinary skill in
the art, e.g., blenders, mixers, mills, homogenizers, dispersers,
dissolvers, etc.

H. In Vitro Artificial Eschar Testing Model

[0121] Enhancement of the enzymatic activity of the compositions was
established by testing the compositions using an in vitro artificial
eschar model as described below and in the publication "Study on the
debridement efficacy of formulated enzymatic wound debriding agents by in
vitro assessment using artificial wound eschar and by an in vivo pig
model", Shi et. al., Wound Repair Regen, 2009, 17(6):853, herein
incorporated by reference. Bovine collagen (Type I), bovine fibrinogen,
and elastin were used to make an Artificial Wound Eschar (AWE) substrate.
Collagen-FITC labeled, elastin-rhodamine, and fibrin-coumarin were the
raw materials used for producing the AWE substrate. To prepare 1 gram of
AWE substrate, 650 mg Collagen-FITC and 100 mg each of elastin-rhodamine
and fibrin-coumarin were weighed into a 50 mL tube and homogenized in 10
mL of Tris buffer saline. In a separate tube, 10 mL of fibrinogen
solution was prepared at 15 mg/mL with Tris buffer saline. The two
solutions were combined and thoroughly mixed. A thrombin solution (0.25
mL at 50 U/mL) was added, quickly mixed, and the solution was poured into
a Petri dish containing a 90 mm nonreactive membrane filter. As a result
of the thrombin-induced fibrinogen polymerization, the material began to
form a soft sheet on top of the membrane filter by clotting the dyed
proteins into a solid matrix. The clotted AWE substrate was allowed to
solidify for 30 minutes and then rinsed with water for 15 minutes to
remove the thrombin. The AWE substrate was further dehydrated to 75%
moisture content in preparation for use.

[0122] With the AWE substrate still attached to the membrane, a 35 mm
diameter piece was punched out using a hole punch. The AWE substrate
punch was placed on the top flat face of a Franz Diffusion Cell System
(Hanson Research, Chatsworth, Calif.), and a TEFLON® sample holder
placed on top. The debriding ointment samples were loaded in the center
of the sample holder, and any excess sample was removed by scraping. The
solution in the receptor cells was Tris buffer at a pH of 7.4 for samples
containing collagenase, papain, thermolysin, or trypsin; and was sodium
acetate buffer at a pH of 2 for samples containing pepsin. The solution
in receptor cells was sampled in 1 mL increments at the following sample
collection times: 0, 1, 2, 3, 6, 12, 18 and 24 hours. Once finished, the
samples were analyzed by fluorescence measurement of FITC dye at 485 nm
(excitation wavelength) and 520 nm (emission wavelength) to determine the
digestion of collagen (collagenolysis) reported in mg/ml.

I. In-Vitro Physical Enzyme Release Test

[0123] The release of enzyme from the compositions was determined by a
Franz cell diffusion study using PVDF (0.45 micron) filters. This study
was performed at 35° C. and lasted for 6 hours. The solution
samples in the receptor cells were subjected to a total protein analysis.

[0124] The protein concentration was determined by a BCA assay (Peirce)
while the same collagenase was used as the reference standard. The
details are described as follows.

[0125] The BCA Protein Assay combines the well-known reduction of
Cu2+ to Cu1+ by protein in an alkaline medium with the highly
sensitive and selective colorimetric detection of the cuprous cation
(Cu1+) by bicinchoninic acid. The first step is the chelation of
copper with protein in an alkaline environment to form a blue-colored
complex. In this reaction, known as the biuret reaction, peptides
containing three or more amino acid residues form a colored chelate
complex with cupric ions in an alkaline environment containing sodium
potassium tartrate. This became known as the biuret reaction because a
similar complex forms with the organic compound biuret
(NH2--CO--NH--CO--NH2) and the cupric ion. Biuret, a product of
excess urea and heat, reacts with copper to form a light blue
tetradentate complex. In the second step of the color development
reaction, BCA, a highly sensitive and selective colorimetric detection
reagent reacts with the cuprous cation (Cu1+) that was formed in
step 1. The purple-colored reaction product is formed by the chelation of
two molecules of BCA with one cuprous ion. The BCA/copper complex is
water-soluble and exhibits a strong linear absorbance at 562 nm with
increasing protein concentrations. The purple color may be measured at
any wavelength between 550 nm and 570 nm with minimal (less than 10%)
loss of signal. See the following reference herein incorporated by
reference: Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,
Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson,
B. J. and Klenk, D. C. (1985). Measurement of protein using bicinchoninic
acid. Anal. Biochem. 150, 76-85.

EXAMPLES

[0126] The following examples are included to demonstrate certain
non-limiting aspects of the invention. It should be appreciated by those
of skill in the art that the techniques disclosed in the examples which
follow represent techniques discovered by the applicants to function well
in the practice of the invention. However, those of skill in the art
should, in light of the present disclosure, appreciate that many changes
can be made in the specific embodiments which are disclosed and still
obtain a like or similar result without departing from the spirit and
scope of the invention.

Example 1

Dispersions of Collagenase/PEG 400 in Petrolatum

[0127] The dispersions in TABLE 1 were prepared with varying
concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in
Petrolatum.

[0128] The enzymatic debridement activity of each dispersion was
determined by the in-vitro artificial eschar model described above and
the results plotted in FIG. 1. As can be seen by the results in FIG. 1,
the optimum amount of PEG-400 based on the 24 hour curve is about 20% w/w
PEG-400.

Example 2

Dispersions of Collagenase/PEG 600 in Petrolatum

[0129] The dispersions in TABLE 2 were prepared with varying
concentrations of Polyethylene Glycol 600 (PEG-600) dispersed in
Petrolatum.

[0130] The enzymatic debridement activity of each dispersion was
determined by the in-vitro artificial eschar model described above. The
results are plotted in FIG. 2. As can be seen by the results in FIG. 2,
the optimum amount of PEG-600 based on the 24 hour curve is about 30% w/w
PEG-600.

Example 3

Dispersions of Collagenase/Poloxamer 124 in Petrolatum

[0131] The dispersions in TABLE 3 were prepared with varying
concentrations of Poloxamer 124 dispersed in Petrolatum.

[0132] The enzymatic debridement activity of each dispersion was
determined by the in-vitro artificial eschar model described above. The
results are plotted in FIG. 3. As can be seen by the results in FIG. 3,
the optimum amount of Poloxamer 124 based on the 24 hour curve is about
30% w/w Poloxamer 124.

Example 4

Dispersions of Trypsin/PEG 400 in Petrolatum

[0133] The dispersions in TABLE 4 were prepared with varying
concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in
Petrolatum.

[0134] The enzymatic debridement activity of each dispersion was
determined by the in-vitro artificial eschar model described above. The
results are plotted in FIG. 4. As can be seen by the results in FIG. 4,
the optimum amount of PEG-400 based on the 24 hour curve is about 14% w/w
PEG-400.

Example 5

Dispersions of Papain/PEG 400 in Petrolatum

[0135] The dispersions in TABLE 5 were prepared with varying
concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in
Petrolatum.

[0136] The enzymatic debridement activity of each dispersion was
determined by the in-vitro artificial eschar model described above. The
results are plotted in FIG. 5. As can be seen by the results in FIG. 5,
the optimum amount of PEG-400 based on the 24 hour curve is about 29% w/w
PEG-400.

Example 6

Dispersions of Thermolysin/PEG 400 in Petrolatum

[0137] The dispersions in TABLE 6 were prepared with varying
concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in
Petrolatum.

[0138] The enzymatic debridement activity of each dispersion was
determined by the in-vitro artificial eschar model described above. The
results are plotted in FIG. 6. As can be seen by the results in FIG. 6,
the optimum amount of PEG-400 based on the 24 hour curve is about 29% w/w
PEG-400.

Example 7

Dispersions of Pepsin/PEG 400 in Petrolatum

[0139] The dispersions in TABLE 7 were prepared with varying
concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in
Petrolatum.

[0140] The enzymatic debridement activity of each dispersion was
determined by the in-vitro artificial eschar model described above. The
results are plotted in FIG. 7. As can be seen by the results in FIG. 7,
the optimum amount of PEG-400 based on the 24 hour curve is about 58% w/w
PEG-400.

Example 8

Dispersions of Collagenase/PEG 400 in Petrolatum for Physical Release of
Enzyme

[0141] The dispersions in TABLE 8 were prepared with varying
concentrations of Polyethylene Glycol 400 (PEG-400) dispersed in
Petrolatum.

[0142] The physical release of enzyme was determined by the In-vitro
Physical Enzyme Release Test as described above. The results are plotted
in FIG. 8. As can be seen by the results in FIG. 8, the physical release
of collagenase generally increased as the concentration of PEG-400 in the
dispersion increased with the highest release at 100% and the lowest
release at 0% PEG-400.

[0143] As can be seen by the results shown herein, the physical enzyme
release profile of the dispersions as a function of increased
concentration of hydrophilic polyol does not correlate to the enzymatic
activity profile of the enzyme as a function of increased concentration
of hydrophilic polyol.

Example 9

Stability and Efficacy Data

[0144] FIG. 9 provides data comparing the stability of collagenase in a
dispersion of the present invention ("30% PEG in WP dispersion") and an
oil-in-water emulsion ("Aqueous cream"). These data suggest that
collagenase was more stable in the 30% PEG in WP dispersion when compared
to the Aqueous cream. Tables 9-10 provide descriptions of the 30% PEG in
WP dispersion and Aqueous cream formulations.

TABLE-US-00009
TABLE 9
(30% PEG in WP dispersion)*
Ingredients wt %
PEG-600 30.059774
Poloxamer-407 1.5078044
White Petrolatum 68.309516
Collagenase 0.1228163
TOTAL 100
*PEG in WP dispersion was prepared as follows: (A) Active Phase: (1) 9.71
grams of PEG-600 and 0.2361 grams of collagenase were mixed for 20
minutes at room temperature (20-25° C.) for 45 min. (B) Main
Phase: (1) 102.784 grams of white petrolatum, 37.65 grams of PEG-600, and
2.27 grams of poloxamer-407 were mixed at 70° C. until uniform;
(2) the mixture was cooled to 40-45° C. Added 7.79 grams of the
Active Phase was added to the Main Phase followed by stirring for 30
minutes or until homogenous mixture obtained.

[0145] FIG. 10 provides data comparing enzyme debridement efficacy in
eschar removal in pig burn wounds of a dispersion of the present
invention ("PEG-in-White Petrolatum"--Table 11) to the following three
formulations: (1) an Aqueous cream--Table 12; (2) SANTYL®
("Commercial product", which is a mixture of collagenase and white
petrolatum); and a hydrogel formulation--Table 13. The burn wounds were
created on pigs and hard eschars formed after several days. Formulation
was applied to the hard eschars one a day for two weeks. Only fully
debrided wounds were counted as "complete debridement." There were a
total of 20 wounds per treatment.

TABLE-US-00011
TABLE 11
(PEG-in-White Petrolatum)*
Ingredients wt %
Poloxamer-407 0.99891551
White Petrolatum 78.7544989
Thermolysin 0.20168104
PEG-600 20.0449046
TOTAL 100
*PEG-in-White Petrolatum was prepared as follows: (A) Active Phase: (1)
32.67 grams of PEG-600 and 1.63 grams of Poloxamer-407 were homogenized
at 70° C. until mixture was clear; (2) mixture was cooled to about
35° C.; and (3) thermolysin was and mixed for at least 30 min..
(B) Main Phase: (1) 236.52 grams of white petrolatum, 30.05 grams of
PEG-600, and 1.5 grams of poloxamer-407 were homogenized at 70°
C.; and (2) mixture was cooled to about 35° C. The Active Phase
(B) was added to the Main Phase (B) and mixed at room temperature
(20-25° C.) for 45 min.